Our research is focused on the mechanisms by which cellular proteins are selected for degradation and the structure/function relationships of the ATP-dependent proteases that degrade them. The ATP-dependent Lon and Clp proteases are found in all organisms, where they serve to modulate the levels of important regulatory proteins and contribute to protein quality control pathways by eliminating damaged proteins. The ATP-dependent proteases are high molecular weight complexes of a molecular chaperone tightly associated with a protease. Reconstruction of electron microscopic images of ClpAP has provided a structural model which helps explain its action and serves as a paradigm for other ATP- dependent proteases. ClpAP is composed 2 seven-membered rings of ClpP flanked on each side by a six-membered ring of ClpA. A large aqueous chamber containing the proteolytic active sites is enclosed by the rings of ClpP. The ClpA subunits enclose another aqueous chamber which may be the site where protein substrates are unfolded prior to translocation to the active site chamber in ClpP. ClpA has two structural distinct ATPase domains in each subunit. Studies with the ClpA mutant, K220V, have shown that the N-terminal ATPase domain (domain I) is required for chaperone activity. Binding of ClpP has allosteric effects on ClpA and can restore the ATPase and chaperone activities to some domain I mutants. In studies done in collaboration with Sue Wickner, NCI, we have shown that substrates bound to ClpAP can be degraded or released in a remodeled state. Thus, the same initial binding chamber is used for both refolding and for translocation to the protease. ClpA and ClpP exchange studies were conducted to measure the half-life of ClpAP complexes during catalysis. Our results show that the complex is stable during multiple rounds of substrate unfolding and degradation, thus showing that proteins interact with the assembled complex and can enter the unfolding and degradation chambers by translocation from external binding sites. Electron microscopic images confirm the model derived from kinetic studies. Studies are underway to dissect the functional domains of a distinct family of ATP-dependent proteases represented by E. coli Lon protease. The Lon ATPase and proteolytic functions lie within a single polypeptide chain. Molecular weight measurements made by ultracentrifugation and by scanning transmission electron microscopy indicate that Lon exists in hexameric and dodecameric forms, with the larger species stabilized by nucleotide binding. Limited proteolysis identified three distinct domains- an N- terminal domain, a central ATPase domain which is stabilized by nucleotide binding, and a C-terminal proteolytic domain which appears to have a limited peptidase activity on its own. Molecular weights of partial cleavage products suggest that the oligomerization domain of Lon lies within the central ATPase region. Electron micrographs of Lon indicate an elongated particle with a pseudo-two fold symmetry and micrographs of sub-oligomers reveal structures with a notched-ring-like appearance. Interactions between the functional domains of Lon may be analogous to those seen with the Clp proteases, suggesting that there is an underlying similarity in architecture for all ATP-dependent proteases. - ATPase, chaperone, post-translational regulation, proteolysis, Clp, protein folding, protein stability, - Neither Human Subjects nor Human Tissues